We have developed physical techniques, namely electron spin resonance spectroscopy (ESR) of spin labels and whole cell isoelectric focusing, to monitor changes in three separate zones of the plasma membrane of ram sperm. All three zones of the plasma membrane differ between testicular and cauda epididymal sperm, but the site(s) within the epididymis where these changes occur and their physiological significance are unknown. The proposed studies will define the nature of sperm membrane changes, identify the site where these changes occur within the epididymis and contribute to understanding the process that induces these maturational changes of the plasma membrane. The integrity of the phospholipid bilayer zone of the plasma membrane and the charge density at the phospholipid-water interface zone will be determined by ESR. The charge of the glycocalyx zone will be evaluated by whole cell isoelectric focusing. These observations, plus light and electron microscopic examinations of sperm after treatment with lectins and iron sol, will be used to determine (a) the location within the epididymis where these maturation-associated changes in sperm membranes occur and (b) if similar changes in membranes can be induced in sperm retained by ligatures at a point in the epididymis proximal to where the membrane changes usually occur. Plasma membranes will be isolated from testicular, cauda epididymal and ejaculated sperm. Protein and lipid constituents of the membranes will be compared. The spectral characteristics of spin labels added to membrane vesicles will be determined to aid in the interpretation of data obtained for intact sperm. Finally, sperm will be incubated in vitro with enzymes to modify the composition of the lipid bilayer or the glycocalyx. The ultimate goal of these incubations is to modify the plasma membrane of testicular sperm so that it is similar to that of cauda epididymal sperm and then reverse the process.